Ophuirid Ophiocomina Nigra HLA-E Gene Synthesis in PUC-GW-KAN Plasmid

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We operate according to the following method [2]. It was resumed in 4 parts: 1. Synthesis of oligonucleotides with overlapping segments in sense and antisense direction. 2. Assembly of the oligonucleotides into a double stranded DNA, using a poly chain assembly method (PCA). 3. For larger constructs, the sequence is split into smaller, intermediate fragments, to facilitate synthesis. Once the intermediated fragments have been obtained with correct sequence, they are assembled into the full-length sequence. 4. Cloning into the linearized vector by either recombination or ligation-based cloning, mostly performed within the same step as full-length sequence assembly. Regarding the restriction site, which was used for cloning, construct was cloned into vector pUC-GW by using the unique EcoRV restriction site. Please find below the primers used for sequencing.

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